Phycobiliprotein-linker peptide complex fluorescent tracer and methods of using the same

ABSTRACT

The use of a phycobiliprotein-linker peptide complex as a fluorescent tracer in a fluorescent method of detecting and/or determining an analyte in a medium in which it may be present, is disclosed. Fluorescent conjugates consisting of said complex covalently bonded to one of the elements of a ligand/receptor specific binding pair, are also disclosed.

The invention relates to the use of a phycobiliprotein-linker peptidecomplex as a fluorescent tracer, notably in a fluorescent method ofdetecting and/or determining an analyte in a medium in which it may bepresent, as well as to the fluorescent conjugates constituted of saidcomplex covalently bound to one of the elements of a ligand/receptorspecific binding pair.

The use of immunological determinations in qualitative and quantitativeanalysis of compounds in biological fluids is currently well known.

Amongst the existing techniques, the determinations by fluorimetry havebecome of increasing importance.

In fact, they possess a certain number of advantages amongst which arethe sensitivity, the speed of measurement, the stability and theinnocuousness of the reagents labelled by fluorescent compounds, and therelatively low cost.

The phycobiliproteins are constituents of the phycobilisome of variousbacteria, algae or cryptomonads and are in general of four types: thephycocyanines, the phycoerythrins, the phycoerythrocyanines and theallophycocyanines.

The proteins are made up of α and β sub-units, and sometimes γsub-units, each one having one or more fluorophores, and are isolatedmainly as trimers or hexamers.

Some of them are used as fluorescent labelling compounds by virtue ofthe many advantages they have in terms of quantum yield, absorptionbands, stability and solubility (V.Oi et al, J. Cell Biology 1982, 93,981).

Schematically, phycobilisomes are made up of two parts:

the core made up of cylindrical elements constituted byallophycocyanines; and

rods, made up of cylindrical elements fixed to the c(ore, said elementsbeing constituted by phycoerythrins and phycocyanines.

The rods and cylinders are made up of an assembly of disks ofphycobiliproteins. These disks are assembled between themselves as wellas the rods to the core and the core to the thylakoid membrane vialinker peptides.

These peptides are named according to their lcicalisation in accordancewith the publication A. Glazer, J. Biol. Chem., 1989, 264, 1-4:

L_(R) for linker peptide in the rod

L_(C) for linker peptide in the core

L_(RC) for linker peptide in the rod-core

L_(CM) for linker peptide in the core-membrane.

The purification of the phycobiliproteins by conventional methods makesit possible to obtain trimeric or hexameric (αβ)₃ or (αβ)₆ complexeswhich are devoid of linker peptides. The trimers have a disk shape of≈30 Å thickness and ≈120 Å diameter.

The use of phycobiliproteins, notably allophycocyanine andphycoerythrin, in immunological determinations by fliiorimetry isdescribed notably in EP 0 174 744 and 0 076 695 as well as in thepatents U.S. Pat. Nos. 4,520,110 and 4,542,104.

It has been found that under certain conditions of purification of thephycobilisomes, it was possible to obtain phycobil-iprotein-linkerpeptide complexes (P. Fuglistaller et al., Biol. Chem. Hoppe-Seyler,1987, 368, 353-367). However, these complexes were hitherto described inthe literature as being relatively unstable and sensitive to proteases(W. Reuter and C. Nickel-Reuter, J. Photochem. Photobiol., B: Biol.,1993, 18, 51-66).

Advantageously, it has now been found that these complexes possessspectroscopic particularities with respect to the phycobiliproteins fora use as a fluorescent tracer.

In fact, such a phycobiliprotein-linker pejptide complex alwayspossesses spectroscopic properties which are different from those of thephycobiliprotein alone, generally with:

an increase in quantum yield

and/or--a shift of the emission wavelength

and/or--a modification of the absorption wavelength

and/or--a modification of the molar absorption coefficient with respectto the phycobiliprotein alone.

These properties can reveal to be particularly interesting during theimplementation of a detection system which uses one or more fluorescenttracers, in which, in addition to the stability of the tracers in themedium, two parameters are of utmost importance:

the quantum yield, which directly influences the limit of detection ofthe system,

the emission wavelength which, during the use of several tracers, is adetermining factor of their choice.

Unexpectedly, it has now been found that:

these phycobiliprotein-linker peptide complexes are stable in solutionand in the presence of sera of various origins containing naturallydifferent proteases,

it is possible to covalently bind these complexes onto various proteinsand antibodies while keeping the fluorescent properties of thecomplexes.

In an advantageous aspect, the phycobiliprotein used according to theinvention is selected from phycoerythrin, phycoerythrocyanine,phycocyanine, allophycocyanine and allophycocyanine B.

In the rest of the description, the following abbreviations will be usedto designate the preferred phycobiliproteins:allophycocyanine=AP,phyco-erythrin=PE, phycoerythrocyanine=PEC, phycocyanine=PC,allophyco-cyanine B=APB

Preferably, the phycobiliprotein-linker peptide complex is extractedfrom a cyanobacterium selected from Mastigoclodus Laminosus,Synechocystis 6701, Synechococcus 6301, Anabaetia iariabilis and Nostocspec.

The linker peptide of the complex used for the purposes of the inventionis preferably selected from the peptides L_(R), L_(C), L_(RC) andL_(CM), such as defined above.

Advantageously, the complexes usable according to the invention aredefined in the following manner, from phycobiliproteins such asdesignated by the abbreviations mentioned above, sub-units α and β andlinker peptides mentioned above:

(α^(PEC), β^(PEC))₆ L_(R), (α^(PEC), β^(PEC))₃ L_(R),

(α^(PC), β^(PC))₆ L_(R), (α^(PC), β^(PC))₆ L_(PC), (α^(PC), β^(PC))₃L_(R),

(α^(AP), β^(AP))₃ L_(C), (α^(APB), α₂ ^(AP), β₃ ^(AP))L_(C) and (α^(AP),β^(AP))₂ L_(CM)

Advantageously, the phycobiliprotein-linker peptide complexes will beused according to the invention in combination with one or moredifferent fluorescent tracers.

The preferred complex for the purposes of the invention is the (α^(AP),β^(AP))₃ L_(C) complex, i.e. a phycobiliprotein-linker peptide complexconstituted of a trimer of α and β sub-units of allophycocyanine and ofthe linker peptide in the core.

The length of the peptides varies according to the species and thedegradation process during the purification. The length is preferablybetween 5000 and 30000.

According to a further aspect. the invention also relates to ahomogeneous fluorescent method of detecting and/or determining ananalyte in a medium in which it may be present, by displaying theproduct of the reaction between the analyte and at least onecorresponding receptor, consisting:

1) in adding, to said medium, a first reagent constituted of at leastone receptor of said analyte,

2) in adding a second reagent selected from the analyte or at least oneof its receptors, one of the two reagents being coupled with afluorescent donor compound constituted by a rare earth cryptate, chelateor macrocyclic complex and the other reagent being coupled with afluorescent acceptor compound, it being possible for the order ofaddition of the reagents to be the reverse order and, after excitationof the mixture by a source of light at the excitation wavelength of thefluorescent donor compound,

3) in measuring the emission signal fluorescent acceptor compound,characterised in that a phycobiliprotein-linker peptide complex such asdefined above is used as fluorescent acceptor compound.

In the present description,

"analyte": defines any substance or group of substances, as well as itsor their analogues, that is (are) desired to be detected and/ordetermined;

"receptor": defines any substance capable of binding specifically to asite of said analyte;

"ligand": defines any substance capable of binding specifically to areceptor.

Rare earth cryptates which can be used in such a determination method aswell as in the excess methods and competitive methcds described beloware notably described in the applications EP 0 180 492, EP 0 232 348, EP0 321 353 or WO90/04791.

Rare earth macrocyclic complexes bearing N-oxy groups are also describedin the application WO93/05049.

These rare earth cryptates and macrocyclic complexes have the advantageof being very stable in proteic and saline media, this property beingparticularly important in the case of homogeneous immunoassays.

A terbium or europium chelate, cryptate or macrocyclic complex bearingN-oxy groups will preferably be used as fluorescent donor compound inthe methods and procedures mentioned in the present description.

According to an advantageous aspect, said method is an excess methodconsisting

1) in adding, to said medium containing the analyte sought after, afirst reagent constituted by at least one receptor of said analyte,coupled with a fluorescent donor compound constituted by a rare earthcryptate, chelate or macrocyclic complex,

2) in adding a second reagent constituted by one or more other receptorsof said analyte, said second reagent being coupled with a fluorescentacceptor compound constituted by a phycobiliprotein-linker peptidecomplex,

3) in incubating said medium after each addition of reagents or afterthe addition of the two reagents,

4) in exciting the resulting medium at the excitation wavelength of thefluorescent donor compound,

5) in measuring the signal emitted by the fluorescent acceptor compound.

One sole receptor of the analyte can notably be used in said methodswhich is coupled either with the fluorescent donor compound, or with thefluorescent acceptor compound.

In another aspect of the invention, said method is a competitive methodconsisting:

1) in adding, to said medium containing the analyte sought after, afirst reagent constituted by a receptor of said analyte, coupled with afluorescent donor compound constituted by a rare earth cryptate, chelateor macrocyclic complex,

2) in adding a second reagent constituted of the analyte coupled with afluorescent acceptor compound constituted by a phycobiliprotein-lirikerpeptide complex,

3) in incubating said medium after each addition of reagents or afterthe addition of the two reagents,

4) in exciting the resulting medium at the excitation wavelength of thefluorescent donor compound,

5) in measuring the signal emitted by the fluorescent acceptor compound.

Advantageously, said homogeneous method using fluorescence to senseand/or determine an analyte in a medium in which it may be present, bydisplaying the product of the reaction between the analyte and at leastone corresponding receptor, is a competitive method consisting:

1) in adding, to said medium containing the analyte sought after, afirst reagent constituted by a receptor of said analyte, said receptorbeing coupled with a fluorescent acceptor compound constituted by aphycobiliprotein-linker peptide complex,

2) in adding a second reagent constituted of the analyte coupled with afluorescent donor compound constituted by a rare earth cryptate orchelate,

3) in incubating said medium either after the addition of each reagent,or after the addition of the two reagents,

4) in exciting the resulting medium at the excitation wavelength of thefluorescent donor compound,

5) in measuring the signal emitted by the fluorescent acceptor compound.

In a preferred aspect of the methods mentioned above, the first reagentand the second reagent are added simultaneously to the medium containingthe analyte sought after.

In a particularly advantageous aspect according to the invention, thefluorescent donor compound used in the methods mentioned above is achelate, a cryptate or a macrocyclic complex of Eu³⁺, and thefluorescent acceptor compound is the (α^(AP), β^(AP))₃ LC complex.

In a further aspect, the invention also relates to the use of aphycobiliprotein-linker peptide complex such as defined above in amethod of amplification of the emission signal of a rare e arth cryptateor chelate used as fluorescent donor compound in a determination byfluorescence, in which a fluorescent acceptor compound is also used,characteris(ed in that the rare earth cryptate or chelate possesses alow overall quantum yield, and in that the quantum yield of radiativedeactivation from the emission level of the rare earth is lower than thequantum yield of the acceptor, said acceptor being constituted by saidphycobiliprotein-linker peptide complex.

In another aspect, the invention also relates to a fluorescent conjugateconstituted of a phycobiliprotein-linker peptide complex covalentlybound to one of the elements of a ligand/receptor specific binding pair.

Said conjugates can particularly be used for sorting the cells and/oranalysing the cell surface in a flow cytometry method, such as notablydescribed in Mel. N.Kronic, J. Immunological Methods, 1986, 92, 1-13.

Protein/protein pairs, protein/DNA pairs or even DNA/DNA pairs maynotably be cited as examples of ligand/receptor specific binding pairs.

Preferably, the phycobiliprotein used according to the invention isselected from phycoerythrin, phycoerythrocyanine, phycocyanine,allophycocyanine and allophycocyanine B and the linker peptide isselected from the peptides L_(R), L_(C), L_(RC) and L_(CM), such asdefined above.

Advantageously, the phycobiliprotein-linker peptide complex is selectedfrom the complexes

(α^(PEC), β^(PEC))₆ L_(R), (α^(PEC), β^(PEC))₃ L_(R),

(α^(PC), β^(PC))₆ L_(R), (α^(PC), β^(PC))₆ L_(RC), (α^(PC), β^(PC))₃L_(R),

(α^(AP), β^(AP))₃ L_(C), (α^(APB), α2^(AP), β₃ ^(AP))L_(C) and (α^(AP),β^(AP))₂ L_(CM),

the (α^(AP), β^(AP))₃ L_(C) complex being preferred.

Preferably, the phycobiliprotein-linker peptide coinplex is extractedfrom a cyanobacterium selected from Mastigoclodus Laminosus,Synechocystis 6701, Synechococcus 6301, Anabaena variabilis and Nostocspec.

Advantageously, the element of the ligand/receptor specific binding pairis a receptor, in particular a cell receptor or an antibody. Saidreceptor can, for example, be avidine or streptavidine.

In another advantageous aspect, said element is a ligand, notably ananalyte. Said ligand can, for example, be a polypeptide, a lectin orbiotin.

EXAMPLE Carcinoembryonic Antigen (CEA) Determination

A homogeneous immunoassay was carried out using the europium cryptate Eutris bipyridine diamine as donor compound prepared as described in theapplication EP 321 353 (examples 3 and 4) coupled to the monoclonalantibody G12 (CIS bio international, France) and either allophycocyanine(Cyanotech, USA), or the (α^(AP), β^(AP))₃ L_(C) complex as acceptorcompound, coupled respectively to the monoclonal antibody G15 (CIS biointernational, France).

The abbreviations used below are the following:

AP=allophycocyanine

DTT=dithiotreitrol

EuTBP=Eu trisbipyridine diamine europium cryptate

BSA=bovine serum albumin

HSA=human serum albumin

IgG=immunoglobulin G

SPDP=N-succinimidyl 3(2-pyridyldithio)propionate

Sulpho-SMCC=sulphosuccinimidyl 4-n-maleimidomethyl)cyclohexane

1) PREPARATION OF THE IgG G15-AP CONJUGATES

a) Activation of AP by sulpho-SMCC

AP (3 mg), commercially available in a precipitated form in a 60%ammonium sulphate solution, is centrifuged. After removal of thesupernatant, the residue is taken up with 250 μl of 100 mM phosphatebuffer, pH 7.0, then filtered at 0.8 μm in order to remove any particlesin suspension.

The filtrate is purified by exclusion chromatography on a superfine G25column (Pharmacia, Sweden) in the same buffer. The conce:ntration of APeluted in the volume of exclusion is determined at 650 nm, in taking anε₆₅₀ nm of 731000M⁻¹ cm⁻¹ into account.

The activation of AP is carried out by adding a solution of sulpho-SMCC,prepared extemporaneously, at the rate of 6.9 mM in a 100 mM phosphatebuffer, pH 7.0, and by allowing the reaction to proceed for an hour atambient temperature with gentle stirring (molar ratio of 15 to 75sulpho-SMCC per AP). The AP-maleimide is then purified on a superfineG25 column in 100 mM phosphate buffer, 5 mM EDTA, pH 6.5, and kept at 4°C. before coupling onto the IgG 3D3.

b) Activation of the IgG G15 by SPDP

Simultaneously, 5 mg of IgG G15 at the rate of 10 mg/ml in 100 mMphosphate buffer, pH 7.0, are activated by the additior, of an SPDPsolution (Pierce, USA) at the rate of 6.4 mM in dioxan in a molar ratioof 7.5 SPDP per IgG G15.

After 35 minutes of activation at ambient temperature, IgG pyridine-2thione is purified on a superfine G25 column in a 100 mM phosphatebuffer, 5 mM EDTA, pH 6.5.

The proteins are concentrated and the 2-pyridyl disulphide groups arereduced with a solution of DTT (Sigma, USA) having a final concentrationof 19 mM for 15 minutes at ambient temperature. The DTT and thepyridine-2-thione are removed by purification on a superfine G25 columnin 100 mM phosphate buffer, 5 mM EDTA, pH 6.5. The concentration ofIgG-SH is determined at 280 nm with an ε₂₈₀ nm of 210000 M⁻¹ cm⁻¹.

c) Conjugation of the IgG G15-SH with AP-maleimide

The binding of the thiol groups onto the maleimides is carried out byadding 2.51 mg of activated AP per mg of IgG G15-SH. After 18 hours ofincubation at 4° C. in the dark with gentle stirring, the thiolfunctions which have remained free are blocked by the addition of a 100mM solution of N-methyl maleimide (Sigma, USA) having a finalconcentration of 20 mM, for one hour at ambient temperature.

The reaction medium is purified by gel filtration on a semi-preparativeTSK G3000SW column (Beckmann, USA) in 100 mM phosphate buffer pH 7.0.

The AP and IgG G 15 concentrations in the purified conjugate, eluted inthe first peak, are determined by the absorptions at 280 nm and 650 nm,according to the following calculation:

[AP]_(Mole/1) =A₆₅₀ nM /10000

[IgG]_(Mole/1) =(A₂₈₀ nm -A'₂₈₀ nm)/210000

with A'₂₈₀ nm being the contribution, at this wavelength, ofAP-maleimide, determined above (paragraph 1-a)).

HSA is added at 1 g/l to the conjugate which is then taken as aliquotsand then frozen at -20° C.

2) PREPARATION OF THE CONJUGATES IgG G15-(α^(AP), β^(AP))₃ L_(C)

a) Activation of the (α^(AP), β^(AP))₃ L_(C) complex by sulphio-SMCC.

The (α^(AP), β^(AP))₃ L_(C) complex is obtained from the algaMasligocladus Laminosus. After extraction of phycobilisomes, theprotein-linkcr peptide complexes are purified according to P.Fuglistaller et al. Biol. Chem. Hoppe Seyler 1986, 367, 601-614.

The complexes have the following properties:

ε650=1076000 M⁻¹ cm⁻¹.

DO₆₅₀ /DO₆₂₀ =2.2.

To 1 ml of (α^(AP), β^(AP))₃ L_(C) complex at 3 mg/ml in 100 mMphosphate buffer pH 7.0, is added 19.5 μl of a solution of sulpho SMCC(Pierce at 30 mmole/l in the same buffer. Incubation is carried out for30 minutes at 30° C. The product of the reaction is purified on a G25 HRcolumn 10×10 flow rate 2 ml/min. The fraction eluted in the dead volumeis recovered (V=1.7 ml). The (α^(AP), β^(AP))L_(C) maleimide complexconcentration is 1.4 mg/ml.

b) Activation of the IgG G15 by SPDP

The activation is carried out as indicated in point 1, b) above.

c) Conjugation of the IgG G15-SH with the (α^(AP), β^(AP))₃ L_(C)-maleimide complex.

1.7 ml of the solution obtained in point 2, a is placed in contact with1 ml of the solution of IgG G15-SH at 0.9 mg/ml obtained above.

After incubation for one night at 4° C. with stirrirng with a rollerstirrer, the conjugate is purified on a semi-preparative TSK 4000 column(Beckmann, USA) with a flow rate of 4 ml/minute in 100 mM phosphatebuffer, pH 7. The fractions of 48 to 64 ml are combined and concentratedon an AMICON cone. The (α^(AP), β^(AP))₃ L_(C) complex concentration inmg/ml is given by the formula DO650/10.76.

The concentration of IgG in mg/ml is given by the formula ##EQU1##

The conjugate is at 120 μl/ml IgG with a molar ratio of 1.6 (α^(AP),β^(AP))₃ L_(C) complex/IgG DO₆₅₀ /620=2.2.

3) PREPARATION OF THE CONJUGATES IgB G12-Eu TBP

The preparation of IgG G12-SH is carried out according to the protocoldescribed above for the G15 3D3 but by varying the molar raijo from 4 to16 SPDP per IgG G12.

To 5 mg (5 10⁻⁶ moles) of Eu TBP is added a 25 mM solution ofsulpho-SMCC in 20 mM phosphate buffer, dimethylformamide 10% (v/v pH 7.0in a proportion of 2.5 moles of activator per mole of Eu TBP.

After 45 minutes' activation at ambient temperature, the reaction mediumis filtered at 0.8 μm in order to remove the precipitate which mighthave formed. The undesirable reaction products (sulpho-SMCC,N-hydroxy-succinimide, (N-maleimidomethyl)carboxylic acid) are removedby ion exchange chromatography on a Mono Q column (Pharnacia, Sweden) in20 mM phosphate buffer, 10% dimethylformamide (v/v), pH 7.0, under NaClshock. The Eu TBP maleimide concentration is determined at 307 nm withan ε₃₀₇ nm of 25000 M⁻¹ cm⁻¹ as well as the ratio A₃₀₇ nm /A₂₈₀ nm.

Similar to that described above, the maleimide functions are allowed toreact with the thiol functions bound to the antibody in molarproportions varying from 10 to 30 Eu TBP maleimide per IgG G12-SH.

After 18 hours of incubation at 4° C. and blockage of the thiol groups(eventually remained free) by N-methylmaleimide, the non-coupled Eu TBPis removed by a dialysis in 100 mM phosphate buffer, pH 7.0, at 4° C.until depletion (no more fluorescence in the dialysis bath).

The properties of the conjugate are determined by its absorptions at 307nm and 280 nm by using the following values in taking into account theactual absorption of the cryptate, determined by the ratio A₃₀₇ nm /A₂₈₀nm.

Eu TBP-maleimide:

ε₃₀₇ nm =25000 M⁻¹ cm⁻¹

A₃₀₇ nm /A₂₈₀ nm : determined experimentally.

IgG G12-SH:

ε₂₈₀ nm =210000 M⁻¹ cm⁻¹

A₃₀₇ nm =0 M⁻¹ cm⁻¹.

4) APPLICATION TO THE CEA DETERMINATION

The G12-Eu TBP, G15-AP and G15-(α^(AP), β^(AP))₃ L_(C) complexconjugates are diluted in a 100 mM phosphate buffer, pH 6, BSA 1 g/l, KF400 mM.

Successively, in polystyrene microplates (Dynatech, USA), are added:

100 μl of standard solution (serum without CEA) or 100 μl of sample tobe tested

100 μl of G12-Eu TBP conjugate at 0.5 μg/ml

100 μl of G15-AP conjugate at 5 μg/ml or 100 μl of G15-(α^(AP), β^(AP))₃L_(C) complex conjugate.

After incubation for 3 hours at 37° C., the reading is taken with theaid of a laser prototype fluorimeter which is described below:

A nitrogen pulsed laser (LASER SCIENCE INC., model LS1-337ND) is used asexcitation source (wavelength at 337.1 nm). The duration of the pulsesis specified at 3 nanoseconds and is repeated under a frequercy of 10Hertz. The beam passes through a filter (CORNING) in order to remove anyparasite light with an excitation other than 337 nm.

After having entered in the measurement chamber, the beam is reflectedby a dichroic filter, placed at 45 degrees, which has the property ofreflecting the ultraviolets and being able to transmit visible light.

The beam reflected by the dichroic filter is focused on the measurementwells of a microplate by a lens of molten silica. The emission offluorescence is collected according to a solid angle of 20 degrees,collimated by the same lens, and passes directly through the dichroicfilter (fluorescence in visible light).

An interference filter, the characteristics of which are definedaccording to the wavelength of fluorescence to be detected, makes itpossible to remove the light which may parasite the signal, whoseintensity is then measured by a photomultiplier (HAMAMATSU R2949).

The photon counter used is an SR-400 (STANFORD RESEARCH SYSTEMS), whoseoperations and synchronisation with the laser are controlled by acomputer of the type IBM PC-AT via an RS 232 exit. The pulsesoriginating from the photomultiplier are recorded during a time window(t_(g)) and after a delay (t_(d)) determined with the proviso that theybe greater than a discriminating level selected by the photon counter inorder to optimise the signal/noise ratio of the photomultiplier.

An X-Y table, piloted by IBM PC-AT, allows for the various positions ofthe measurement microplate by the stepwise motors, including the loadingmanoeuvres, positioning under the exciting beam, automatic sequentialreading of the 96 wells, and exiting.

The fluorescence emitted by the G15-AP or G15-(α^(AP), β^(AP))₃ L_(C)complex conjugate is measured with the aid of the prototype fluorimeterequipped with a filter at 665 nm of 10 nm length at mid-height, for 400μs and with a delay of 50 μs.

The results are given in the Table below, expressed in Δcpscorresponding to the difference between the signal emitted by the sampleat 665 nm with respect to the standard solution (serum without CEA).

    ______________________________________                                        CEA ng/ml  AP Δcps                                                                          (α.sup.AP, β.sup.AP).sub.3 L.sub.C complex                         Δcps                                                ______________________________________                                        4.4         251      388                                                        15.9  966 1670                                                                118 6427 9804                                                                 236 9436 16232                                                              ______________________________________                                    

The results show that the signal measured is larger for thephycobiliprotein-linker protein complex with respect to thephycobiliprotein alone.

Furthermore, it is noted that the ratio DO₆₅₀ /DO₆₂₀ =2.2 whichcharacterises said complex is constant within the limit of measurementerrors for all the steps of the preparation of the conjugate, and verydifferent from that of the AP (≈1,45).

This demonstrates the stability of the phycobiliprotein-linker peptidecomplex throughout the steps of purification and coupling which arecarried out without any particular precautions with respect to that ofthe G15 AP conjugate.

I claim:
 1. A fluorescent method of detecting and/or determining ananalyte in a medium in which it may be present, characterised in that aphycobiliprotcin-linker peptide complex is used as a fluorescent tracer.2. The method according to claim 1, characterised in that thephycobiliprotein of said phycobiliprotein-linker peptide complex isselected from phycocrythrin, phycoerythrocyanine, phycocyanine,allophycocyanine and allophycccyanine B.
 3. The method according toclaim 1 characterised in that the phycobiliprotein-linker peptidecomplex is extracted from a cyanobacterium selected from MastigoclodusLaminosus, Synechocystis 6701, Synechococcus, 6301, Anabaena variabilisand Nostoc spec.
 4. The method according to claim 1, characterised inthat the linker peptide of said phycobiliprotein-linker peptide complexis selected from the peptides L_(R), L_(C), L_(RC) and L_(CM).
 5. Themethod according to claim 1, characterised in that thephycobiliprotein-linker peptide complex is selected from thecomplexes(α^(PEC), β^(PEC))₆ L_(R), (α^(PEC), β^(PEC))₃ L_(R), (α^(PC),β^(PC))₆ L_(R), (α^(PC), β^(PC))₆ L_(RC), (α^(PC), β^(PC))₃ L_(R),(α^(AP), β^(AP))₃ L_(C), (α^(APB), [α2]α₂ ^(AP), [B3]β₃ ^(AP))L_(C) and(α^(AP), β^(AP))₂ L_(CM).
 6. A homogenous fluorescent method ofdetecting and/or determining an analyte in a medium in which it may bepresent, by displaying the product of the reaction between the analyteand at least one corresponding receptor, consisting of:1) adding to saidmedium a first reagent constituted of at least one receptor of saidanalyte, 2) adding a second reagent selected from the analyte or atleast one of its receptors, one of said first reagent or said secondreagent coupled with a flourescent donor compound constituted by a rareearth cryptate, chelate or macrocyclic complex and other reagent beingcoupled with a fluorescent acceptor compound, it being possible for theorder of addition of the reagents to be the reverse order and, afterexcitation of the mixture by a source of light at the excitationwavelength of the fluorescent donor compound, 3) measuring the emissionsignal of the fluorescent acceptor compould, and 4) correlating themeasured emission signal of the fluorescent acceptor compound to thepresence and/or amount of analyte, characterised in that aphycobiliprotein-linker peptide complex is used as a fluorescentacceptor compound.
 7. The method according to claim 6 which consists ofan excess method, characterised in that it consists of:1) adding to saidmedium containing the analyte sought after a first reagent constitutedby at least one receptor of said analyte coupled with a fluorescentdonor compound constituted by a rare earth cryptate, chelate ormacrocyclic complex, 2) adding a second reagerit constituted by one ormore other receptors of said analyte, said second reagent being coupledwith a fluorescent acceptor compound constituted by aphycobiliprotein-linker peptide complex, 3) incubating said medium aftereach addition of reagents or after the addition of the second reagent,4) exciting the resulting medium at the excitation wavelength of thefluorescent donor compound, 5) measuring the signal emitted by thefluorescent acceptor compound.
 8. The method according to claim 6consisting of a competitive method, characterised in that it consistsof:1) adding to said medium containing the analyte sought after a firstreagent constituted by a receptor of said analyte, coupled with afluorescent donor compound constituted by a rare earth cryptate, chelateor macrocyclic complex, 2) adding a second reagent constituted of theanalyte coupled with a fluorescent acceptor compound constituted by aphycobiliprotein-linker peptide complex, 3) incubating said medium aftereach addition of reagents or after the addition of the second reagent,4) exciting the resulting medium at the excitation wavelength of thefluorescent donor compound, 5) measuring the signal emitted by thefluorescent acceptor compound.
 9. The method according to claim 6consisting of a competitive method, characterised in that it consistsof:1) adding to said medium containing the analyte sought after a firstreagent constituted by a receptor of said analyte, said receptor beingcoupled with a fluorescent acceptor compound constituted by aphycobiliprotein-linker peptide complex, 2) adding a second reagentconstituted of the analyte coupled with a fluorescent donor compoundconstituted by a rare earth cryptate or chelate, 3) incubating saidmedium either after the addition of each reagent, or after the additionof the second reagent, 4) exciting the resulting medium at theexcitation wavelength of he fluorescent donor compound, 5) measuring thesignal emitted by the fluorescent acceptor compound.
 10. The methodaccording to any one of claims 6 to 9, characterised in that the firstreagent and the second reagent are added simultaneously to the mediumcontaining the analyte sought after.
 11. The method according to any oneof claims 6 or 7, characterised in that one sole receptor of the analyteis used which is coupled either with the fluorescent donor compound, orwith the fluorescent acceptor compound.
 12. The method according to anyone of claims 6 to 9, characterised in that the fluorescent donorcompound is a terbium or europium chelate, cryptate or macrocycliccomplex.
 13. The method according to any one of claims 6 to 9characterised in that the fluorescent donor compound is an Eu³⁺ chelate,cryptate or macrocyclic complex and the fluorescent acceptor compound isthe (α^(AP), β^(AP))₃ L_(C) complex.
 14. A method of amplification ofthe emission signal of a rare earth cryptate or chelate used asfluorescent donor compound in a fluorescent assay according to claim 1,in which a fluorescent acceptor compound is also used, characterised inthat the rare earth clyptate or chelate possesses a low overall quantumyield, and in that the quantum yield of radiative deactivation from theemission level of the rare earth is lower than the quantum yield of theacceptor being constituted by a phycobiliprotein-linker peptide complex.15. The method according to claim 1, characterised in that thephycobiliprotein-linker peptide complex is used in combination with oneor more different fluorescent tracers.
 16. A fluorescent conjugateconstituted of a phycobiliprotein-linker peptide complex covalentlybound to one of the elements of a ligand/receptor specific binding pair.17. The conjugate according to claim 16, characterized in that thephycobiliprotein of said phycobiliprotein-linker peptide complex isselected from phycoerythrin, phycoerythrocyanine, phycocyanine,allophycocyanine and allophycocyanine B.
 18. The conjugate according toclaim 16 or 17, characterized in that the linker peptide of saidphycobiliprotcin-linkcr peptide complex is selected from the peptidesL_(R), L_(C), L_(RC) and L_(CM).
 19. The conjugate according to one ofclaims 16 or 17, characterised in that the phycobiliprotein-linkerpeptide complex is selected from the complexes(α^(PEC), β^(PEC))₆ L_(R),(α^(PEC), β^(PEC))₃ L_(R), (α^(PC), β^(Pc))₆ L_(R), (α^(PC), β^(PC))₆L_(RC), (α^(PC), β^(PC))₃ L_(R), (α^(AP), β^(AP))₃ L_(C), (α^(APB),[α2]α₂ ^(AP) ; [B3]β₃ ^(AP))L_(C) and (α^(AP), β^(AP))₂ L_(CM).
 20. Theconjugate according to one of claims 16 or 17, characterised in that thephycobiliprotein-linker peptide complex is extracted from ac),anobacterium selectcd from Mastigoclodus Laminosus, Synechocystis6701, Synechococcis 6301, Anabaena variabilis and Nostoc spec.
 21. Theconjugate according to claim 16, characterised in that the element ofthe ligand/receptor specific binding pair is a receptor.
 22. Theconjugate according to claim 16, characterised in that the element ofthe ligand/receptor specific binding pair is a ligand.
 23. A flowcytometry method characterized by the use of the conjugate of claim 16or
 17. 24. The method of claim 6 wherein phycobiliprotein of saidphycobiliprotein-linker peptide complex is selected from phycoerythrin,phycoerythrocyanine, phycocyanine, allophycocyanine and allophycocyanineB.
 25. The method of claim 6 wherein said phycobiliprotein-linkerpeptide complex is extracted from a cyanobacterium selected fromMastigoclodus Laminosus, Synechocystis 6701, Synechococcus, 6301,Anabaena variabilis and Nostoc spec.
 26. The method of claim 6 whereinthe linker peptide of said phycobiliprotein-linker peptide complex isselected from the peptides L_(R), L_(C), L_(RC) and L_(CM).
 27. Themethod of claim 6 wherein said phycobiliprotein-linker peptide complexis selected from the complexes(α^(PEC), β^(PEC))₆ L_(R), (α^(PEC),β^(PEC))₃ L_(R), (α^(PC), β^(PC))₆ L_(R), (α^(PC), β^(PC))₆ L_(RC),(α^(PC), β^(PC))₃ L_(R), (α^(AP), β^(AP))₃ L_(C), (α^(APB), α2^(AP),B3^(AP))L_(C) and (α^(AP), β^(AP))₂ L_(CM).
 28. The conjugate of claim21 wherein said receptor is a cell receptor or an antibody.
 29. Theconjugate of claim 22 wherein said ligand is an analyte.